Arteriosclerosis depressant

ABSTRACT

9-methyl-3-(1H-tetrazol-5-yl)-4H-pyrido 1, 2-a!pyrimidin-4-one represented by the following formula; ##STR1## and the physiologically-acceptable salts thereof showed to have excellent inhibitory effect on the proliferation of vascular smooth muscle cells and are effective to prevent the occurrence of restenosis after the operation of PTCA, and hence the compounds can be useful for the therapeutic and preventive treatment for arteriosclerosis and other diseases whereto the proliferation of vascular smooth muscle cells is directly concerned.

This is a Section 371 Application of PCT/JP95/00277 filed Feb. 24, 1995,which was a filing of Japan Application 6-119146 filed May 31, 1994.

FIELD OF THE INVENTION

The present invention is directed to a remedy for inhibitingarteriosclerosis containing 9-methyl-3-(1H-tetrazol-5-yl)-4H-pyrido1,2-a!pyrimidin-4-one or the physiologically-acceptable salt thereof asan active component.

BACKGROUND ART

Arteriosclerosis is defined in general as a physical state in whicharterial wall locally thickness and deposition of lipids and calciumsalts on the thickened-parts of arterial wall takes place, whereby theelastic fibers of the vascular wall are destroyed and hence theelasticity of blood vessel is lost (see Yakukagaku Daijiten, 2ndEdition, Hirokawa Shoten, 1990), Arteriosclerosis is considered to bethe cause of various diseases. Until today, clofibrates, such asclofibrate, simfibrate and alufibrate, Nicotinic acids, such as nicomoland tocopherol nicotinate, dextran sulfate esters, pantetin,cytosterol-based preparations such as soysterol, anabolic steroids, suchas furazabol and oxandrolone, elastase, pravastatin and the like havebeen developed as therapeutic remedy for arteriosclerosis, however,there have been no remedies which have shown sufficient therapeuticeffect.

It Is reported as follows concerning the mechanism of the proliferationand thickening of arterial wall. Various factors are known which causethe degeneration and ablation of endothelium cells of the arterial wall.Once such degeneration and ablation of endothelium cells is caused,platelets tend to attach to the tissues formed under the endotheliumcells coagulate, then an accelerating factor for the proliferation ofvascular smooth muscle cells is released from alpha-granules.Consequently, the smooth muscle cells migrate toward the intra of thearterial wall and then proliferate there to cause the thickening of thearterial wall. (See Ross, R., Glomset, J. A. :The pathogenesis ofatherosclerosis, N. Engl. J. Med., 295, 369-377, 420-425, 1976. Ross, R.:The pathogenesis of atherosclerosis--an up date, N. Engl. J. Med., 314,488-500, 1986).

Considering the mechanism described above, it is understood thatremedies capable of inhibiting the proliferation of smooth muscle cellscan inhibit the occurrence of arteriosclerosis as well.

Recently, a therapeutic technique called Percutaneous TransluminalCoronary Angioplasty (hereinafter referred to as PTCA) that treatsnarrowed blood vessels by inserting a ballon catheter into the vesselsto widen them, has been widely accepted. However, restenosis of bloodvessels becomes known sometime during the period 3 to 6 months afterPTCA and is caused by the proliferation of smooth muscle cells.Therefore, it is understood that the inhibition of the proliferation ofvascular smooth muscle cells can be an effective means to prevent therestenosis of blood vessels after PTCA.

It is an object of the present invention to provide a substance capableof inhibiting the proliferation of vascular smooth muscle cells.

SUMMARY OF THE INVENTION

The inventors of the present invention investigated substances having aninhibitory effect on the proliferation of vascular smooth muscle cells,and as a result, they found out that9-methyl-3-(1H-tetrazol-5-yl)-4H-pyrido 1,2-a!pyrimidin-4-one(hereinafter referred to as Compound 1) and thephysiologically-acceptable salts thereof, which are known to have aninhibitory effect on allergic action (see Japanese Patent Laid-openedNo. Sho 54-36294 Gazette), have an inhibitory effect on theproliferation of vascular smooth muscle cells that has not been known inthe past, and thereby completed the present invention.

Compound 1, an active ingredient for inhibiting arteriosclerosis of thepresent invention, can be obtained according to the followingpreparation procedure.

Firstly, ethyl 2-cyano-3-(3-methyl-2-pyridylamino)acrylate is reactedwith sodium azide in the presence of aluminium chloride or the like toform a compound with a tetrazol ring. The compound is then subjected tofiltration under acidic conditions to separate Compound 1. Examples ofthe physiologically-acceptable salts of Compound 1, are the potassiumsalt and the sodium salt.

The remedy for inhibiting arteriosclerosis of the present invention canbe administrated orally and parenterally, and the doses of the remedycan be determined depending upon the age, symptoms, body weight and sexof the patients and other factors. In general, adequate dose per day ofthe active component, either Compound 1 or the salts thereof, is in arange of from 1 mg to 5 g, and more preferably from 5 mg to 1 g, for theoral administration, whereas in a range of from 0.2 mg to 1 g, and morepreferably from 1 mg to 300 mg, for the parenteral administration. As tothe direction for use for Compound 1 and the salt thereof, it isadequate to administer the compound 1 to 4 times per day, and morepreferably once or twice per day, as far as within the dose range asspecified above.

The Compound 1 and the salts thereof of the present invention can bepharmaceutically prepared into solid preparations, such as tablets,pills, capsules, powders, fine granules, granules and suppositories, orliquid preparations, such as solutions, medicated syrups, suspensions,emulsions and solutions for injection, by combining either solid orliquid physiologically-acceptable carriers with Compound 1 or the saltthereof. In the case of solid preparations, Compound 1 and the saltthereof may be prepared into enteric coated preparations and sustainedrelease preparations. For the carriers usable for the pharmaceuticalpreparations of Compound 1 and the salt thereof, any carrier beingcommonly used for pharmaceutical preparations can be utilized, and, forexamples, excipients, such as corn starch, dextrin, alpha beta- andgamma-cyclodextrins, glucose, lactose, sucrose, methyl cellulose,hydroxypropyl cellulose, carboxymethyl cellulose calcium, crystallinecellulose, sodium alginate, Witepsol W35, Witepsol E85 and polyvinylalcohol; either binders or disintegrating agents; lubricants, such astalc, stearic acid, magnesium stearate and light anhydrous silicic acid;coating agents, such as shellac, cellulose acetate phthalate,polyvinylacetaldiethylaminoacetate, carboxymethylethyl cellulose,hydroxypropylmethyl cellulose acetate succinate, cellulose hydroxymethylphthalate, and methylmethacrylate methacrylic acid copolymer; solutionadjuvant, such as glycerin, propylene glycol and mannitol; emulsifyingagents, such as polyoxyethylene stearate and polyoxyethylene laurylalcohol ether; and suspending agents, such as acacia andpolyvinylpyrrolidone, can be exemplified. In addition thereto,stabilizing agents, solvents and/or adequate perfumes may be used, ifrequired.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Now, the inhibitory effect on the proliferation of vascular smoothmuscle cells and the result of clinical test is described hereinbelow indetail, when potassium salt of Compound 1 (hereinafter referred to asCompound 1-K) is used as the representative for the remedy forInhibiting arteriosclerosis of the present invention.

EXPERIMENTAL EXAMPLE 1 Inhibitory Effect on Deoxyribonucleic Acid (DNA)Synthesis in Vascular Smooth Muscle Cells

To each well of a plate with 96 wells, cultured smooth muscle cells ofaorta of a rat in an amount of 100,000 cells/well was placed,respectively. After the adhesion of the cells to the wells was made,Compound 1-K solutions in different concentrations were added to eachwell, respectively, and the cells were then incubated for 36 hours inDulbecco's modified-medium added with 10% fetal calf serum. The cellswere then further incubated for 2 hours in bromodeoxyuridine, thenincorporation of bromodeoxyuridine into DNA was measured according toELISA method. The rejective ratios of incorporation of bromodeoxyuridineinto DNA in different concentrations of Compound 1-K solution were shownin Table 1.

                  TABLE 1                                                         ______________________________________                                        INHIBITORY EFFECT ON DEOXYRIBONUCLEIC ACID                                    SYNTHESIS IN VASCULAR SMOOTH MUSCLE CELLS                                                          Ratio of incorporation                                             Concentration (M)                                                                        of BrdU into DNA (%)                                     ______________________________________                                        Control                  100                                                  Compound 1-K                                                                              1 × 10.sup.-8                                                                        106                                                              1 × 10.sup.-7                                                                        94                                                               1 × 10.sup.-6                                                                        86                                                               1 × 10.sup.-5                                                                        67                                                               1 × 10.sup.-4                                                                        66                                                   ______________________________________                                         BrdU: Bromodeoxyuridine                                                  

Compound 1-K showed concentration-dependent Inhibitory effect on DNAsynthesis in the vascular smooth muscle cells at a concentration of1×10⁻⁷ (M) or higher.

EXPERIMENTAL EXAMPLE 2 Inhibitory Effect on Proliferation of VascularSmooth Muscle Cells

To each well of a plate with 6 wells, cultured smooth muscle cells ofaorta of a rat in an amount of 100,000 cells/well was placed,respectively. After the reaching of the cells to sub-confluentcondition, Compound 1-K solutions in different concentrations were addedto each well, respectively, then the cells were incubated for 48 hoursin Dulbecco's modified-medium added with 10% fetal calf serum. Thenumber of the cells per each well was then counted. The number of thecells per well are shown in Table 2.

                  TABLE 2                                                         ______________________________________                                        INHIBITION EFFECT ON PROLIFERATION OF VASCULAR                                SMOOTH MUSCLE CELLS                                                           Concentration (M)                                                                              Number of Cells (× 10.sup.4 /well)                     ______________________________________                                        Control              33.5                                                     Compound 1-K                                                                              1 × 10.sup.-7                                                                    29.8                                                                 1 × 10.sup.-4                                                                    27.8                                                                 1 × 10.sup.-5                                                                    19.8                                                                 1 × 10.sup.-4                                                                    17.8                                                     ______________________________________                                    

Compound 1-K showed inhibitory effect on the proliferation of vascularsmooth muscle cells at a concentration of 1×10⁻⁷ (M) or higher.

EXPERIMENTAL EXAMPLE 3 Clinical Tests

Patients with first elective PTCA were separated at random into a groupto receive the administration of Compound 1-K (P-group: 26 patients, 39lesions) and a group having no administration of Compound 1-K (C-group:25 patients, 31 lesions) to carry out random comparison tests.

Compound 1-K in an amount of 20 mg/day was continuously administrated tothe patients in a period from 2 weeks before the operation of PTCA tillfollow-up angiography, which was carried out in average at 4 monthsafter the operation of PTCA. To all patients in the both groupsdescribed above, 81 mg of aspirin, calcium antagonist and nitrates drugwere also administrated, respectively. The % stenosis was measured byusing video densitometry analyser (Manufactured by PADL), and the casegained more than 20% reduction in % stenosis and less than 50% ofremaining-% stenosis is defined as successful PTCA, whereas the caselost more than 50% of the gain obtained by PTCA or showed more than 50%of remaining-% stenosis is defined as restenosis. It should be notedthat no difference in ratio on the sexes, age, coronary risk factors,symptom types of angina pectoris, member of diseased vessels andbackground of coronary artery disease was recognized between P-group andC-group. The results are shown in Table 3.

                  TABLE 3                                                         ______________________________________                                        CHANGE OF % CORONARY STENOSIS AND RESTENOSIS RATE                                             P-Group C-Group                                               ______________________________________                                        % Stenosis before PTCA (%)                                                                      77.6 ± 11.7                                                                          72.7 ± 8.7                                     % Stenosis after PTCA (%)                                                                       19.7 ± 11.2                                                                          21.5 ± 10.4                                    % Stenosis at Follow-up (%)                                                                     29.6 ± 21.0                                                                          46.8 ± 22.3                                    Restenosis Rate (%)                                                                             12.8%*    43.9%                                             ______________________________________                                         *p < 0.01                                                                

It was confirmed that Compound 1-K has preventive effect on coronaryrestenosis.

Now, it is explained hereinbelow about the pharmaceutical preparationfor Compound 1-K.

EXAMPLE 1 Preparation of Tablets

10.0% by weight of Compound 1-K, 56.0% by weight of lactose, 15.0% byweight of corn starch, 15.0% by weight of crystalline cellulose and 3.0%by weight of hydroxypropyl cellulose were mixed together, and themixture was then subjected to granulation with adding water andsubsequently dried.

After shaping the granules obtained, magnesium stearate in an amount of1.0% by weight was further added to the granules and mixed, then themixture was subjected to shaping under compression to prepare 100mg/tablet weight of tablets.

EXAMPLE 2 Preparation of Capsules

According to a customary procedure, 10.0% by weight of Compound 1-K,65.5% by weight of lactose, 20.0% by weight of corn starch, 3.0% byweight of hydroxypropyl cellulose, 0.5% by weight of light anhydroussilicic acid and 1.0% by weight of magnesium stearate were mixedtogether, and the mixture was then subjected to granulation to form intothe granules. The granules obtained were charged into capsules toprepared 100 mg/capsule weight of capsules.

EXAMPLE 3 Preparation of Granules

10.0% by weight of Compound 1-K, 73.0% by weight of lactose, 10.0% byweight of low substituted hydroxypropyl cellulose, 5.0% by weight ofpolyvinyl pyrrolidone and 2.0% by weight of sodium lauryl sulfate weremixed together, and the mixture was then kneaded with adding water, andsubsequently prepared into granules in cylindrical shape by using aoscillating granulator.

On the basis of the property to show an inhibitory effect on theproliferation of vascular smooth muscle cells, Compound 1 and thephysiologically-acceptable salts thereof of the present invention can beuseful for the therapeutic remedy for arteriosclerosis whereto theproliferation of vascular smooth muscle cells is directly concerned andfor restenosis after the operation of PTCA.

What is claimed is:
 1. A pharmaceutical preparation containing9-methyl-3-(1H-tetrazol-5-yl)-4H-pyrido 1, 2-a!pyrimidin-4-onerepresented by the following chemical formula: ##STR2## Or thephysiologically-acceptable salt thereof in an effective amount to treatarteriosclerosis, and a physiologically acceptable carrier.
 2. Apharmaceutical preparation according to claim 1, in solid form.
 3. Apharmaceutical preparation according to claim 1, in liquid form.
 4. Apharmaceutical preparation according to claim 2, wherein said solid formis suitable for oral administration.
 5. A pharmaceutical preparationaccording to claim 3, wherein said liquid form is suitable forparenteral administration.
 6. A pharmaceutical preparation according toclaim 2, wherein said preparation further comprises one or morecomponents selected from the group consisting of excipients, binders,disintegrating agents, and lubricants, and coating agents.
 7. Apharmaceutical preparation according to claim 3, wherein saidpreparation further comprises one or more components selected from thegroup consisting of solution adjuvants, emulsifying agents andsuspending agents.
 8. A pharmaceutical preparation according to claim 6,wherein said composition is tabletted.
 9. A pharmaceutical preparationaccording to claim 6, wherein said composition is disposed in capsules.